U.S. patent number 3,616,251 [Application Number 04/774,490] was granted by the patent office on 1971-10-26 for test device.
This patent grant is currently assigned to Miles Laboratories, Inc.. Invention is credited to Gianni Linoli, Enzo Sergio Mannucci.
United States Patent |
3,616,251 |
Linoli , et al. |
October 26, 1971 |
TEST DEVICE
Abstract
A test device useful for the detection of a component of a fluid
system comprises a water-resistant carrier, such as an
organoplastic strip, containing, as an integral part thereof, a
test system for the detection of such component. This test device
is produced by incorporating the test system into at least a
partially dissolved portion of the carrier material and then
solidifying such carrier material with the test system included
therein.
Inventors: |
Linoli; Gianni (Lecco,
IT), Mannucci; Enzo Sergio (Calolziocorte,
IT) |
Assignee: |
Miles Laboratories, Inc.
(Elkhart, IN)
|
Family
ID: |
25101411 |
Appl.
No.: |
04/774,490 |
Filed: |
November 8, 1968 |
Current U.S.
Class: |
435/12; 435/16;
435/18; 435/19; 435/21; 435/24; 435/805; 436/163; 436/903;
422/404 |
Current CPC
Class: |
G01N
31/221 (20130101); G01N 33/521 (20130101); C12Q
1/00 (20130101); Y10S 435/805 (20130101); Y10S
436/903 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101); G01N 33/52 (20060101); G01N
31/22 (20060101); G01n 031/14 (); G01n
031/22 () |
Field of
Search: |
;195/13.5R,99,13.5C,127
;23/253TP,23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Monacell; A. Louis
Assistant Examiner: Hoffman; James R.
Claims
What is claimed is:
1. A test device for the detection of a component of a fluid system
which comprises a transparent organoplastic water resistant carrier
selected from the class consisting of cellulose acetate,
polystyrene, ethyl cellulose, cellulose nitrate, celluloid,
polyvinyl chloride, polyethylene and polymethylmethacrylate
containing, as an integral part thereof, a test system selected
from the class consisting of an insoluble polymeric diazonium salt
of an aromatic polyamine bound through an amide linkage to a cation
exchange resin, and an insoluble pH-sensitive reaction product
which undergoes a color change with a change in pH, said reaction
product being selected from the class consisting of (1) the
reaction product between an insoluble polymeric diazonium salt of
an aromatic polyamine bound through an amide linkage to a cation
exchange resin and a suitable coupling compound, (2) the reaction
product between an insoluble diazonium salt of para-aminobenzyl
cellulose and a suitable coupling compound, (3) the reaction
product between a polycarboxylic resin acyl halide and a compound
having at least one free amino or phenolic group, (4) the reaction
product between an aromatic diazonium salt and the reaction product
between a polycarboxylic resin acyl halide and an aromatic
polyamine, and (5) the reaction product between an insoluble
polymeric material having free amino groups and a pH sensitive
compound which undergoes a color change with a change in pH.
2. A test device according to claim 1 wherein the test system
comprises an insoluble polymeric diazonium salt of an aromatic
polyamine bound through an amide linkage to a cation exchange
resin.
3. A test device according to claim 2 wherein the aromatic
polyamine portion of the polymeric diazonium salt is
o-dianisidine.
4. A test device according to claim 2 for the detection and/or
determination of an enzyme wherein the test system also includes a
substrate on which the enzyme can react to liberate a coupling
compound which can then react with the insoluble polymeric
diazonium salt to produce a color change.
5. A test device according to claim 1 wherein the test system is
incorporated into at least a surface portion of the organoplastic
carrier.
6. A test device according to claim 1 wherein the test system
comprises an insoluble PH sensitive reaction product which
undergoes a color change with a change in pH, said reaction product
being selected from the class consisting of (1) the reaction
product between an insoluble polymeric diazonium salt of an
aromatic polyamine bound through an amide linkage to a cation
exchange resin and a suitable coupling compound, (2) the reaction
product between an insoluble diazonium salt of para-aminobenzyl
cellulose and a suitable coupling compound, (3) the reaction
product between a polycarboxylic resin acyl halide and a compound
having at least one free amino or phenolic group, (4) the reaction
product between an aromatic diazonium salt and the reaction product
between a polycarboxylic resin acyl halide and an aromatic
polyamine, and (5) the reaction product between an insoluble
polymeric material having free amino groups and a pH sensitive
compound which undergoes a color change with a change in pH.
7. A test device according to claim 6 wherein the insoluble pH
sensitive reaction product is the reaction product between an
insoluble polymeric diazonium salt of an aromatic polyamine bound
through an amide linkage to a cation exchange resin and a suitable
coupling compound.
8. A test device according to claim 6 for the detection and/or
determination of urea in a fluid wherein the test system includes
urease and the insoluble pH sensitive reaction product between an
insoluble polymeric material having free amino groups and a pH
sensitive compound which undergoes a color change with a change in
pH.
Description
BACKGROUND AND DISCUSSION OF PRIOR ART
Test devices for the detection and/or determination of various
components of fluid systems have been known for many years. For the
most part, these prior art test devices consisted essentially of
bibulous materials, such as filter paper, impregnated with the
reagent components of the test system. These prior art materials
had the main disadvantage that the test system reagent components
were often soluble in the fluid system being tested. This was
especially true for test devices useful for measuring the pH of a
fluid system. It was suggested in the prior art to form selectively
permeable protective barriers on the reagent impregnated bibulous
carriers by coating the carriers with a plastic coating, such as
that obtained from ethyl cellulose. These prior art coated test
devices had the disadvantage of requiring additional processing
steps in their manufacture.
STATEMENT OF THE INVENTION
It has now been found that an improved test device can be prepared
which overcomes the disadvantages of the prior art devices. In
accordance with the present invention, a test device for the
detection of a component of a fluid system is provided which
comprises a water-resistant carrier containing, as an integral part
thereof, a test system for said component. This test device is
prepared by the novel process of incorporating said test system
into at least a partially dissolved portion of water-resistant
carrier material and solidifying said water-resistant carrier
material with the test system included therein. In particular, this
novel test device is preferably prepared by contacting a solidified
portion of a water-resistant carrier material with a mixture of the
test system and a solvent for said carrier material, whereby at
least a surface portion of the solidified carrier material is at
least partially dissolved to allow the test system to be mixed
therein prior to resolidification of the carrier material by
evaporation of the solvent.
The test devices of the present invention can be employed to detect
and/or determine various components of a fluid system. Exemplary of
such component is an enzyme, such as a phosphatase, which is
present in a body fluid, such as serum. This enzyme may be capable
of liberating a coupling compound, such as beta-naphthol, from a
substrate, such as beta-naphthylphosphate. This liberated coupling
compound may then form a reaction product with a chromogen. The
intensity of the resulting color is dependent on the concentration
of the coupling compound and thus is an indication of the amount of
the specific enzyme in the test sample. THe test device for an
enzyme can contain, incorporated in the water-resistant carrier, a
mixture of the substrate and the chromogen. Alternatively, the
substrate can be in a separate composition which is used first to
react with an enzyme-containing sample and then a test device
containing a chromogen is contacted with the resulting coupling
compound-containing reaction mixture. The following is a list of
representative enzymes that can be measured by test devices of the
present invention.
---------------------------------------------------------------------------
Liberated Enzyme Substrate Coupling Compound
__________________________________________________________________________
Phosphatase naphthyl- Beta-naphthol phosphate Phosphatase
Alpha-naphthyl- Alpha-naphthol phosphate Lipase Beta-naphthyl-
Beta-naphthol laurate Glutamic oxalo- Sodium aspartate- Oxaloacetic
acetic transaminase alpha-ketoglutarate acid Leucine amino-
Alpha-leucine-beta Beta-naphthyl- peptidase -naphthylamine amine
Esterase Naphthyl esters Naphthol N-acethylbeta- Naphthyl-beta-
Naphthol glucosaminidase acetyl glucosamine Oxytocinase
Cysteine-meta Meta-anisidine -anisidide
__________________________________________________________________________
A test device of the present invention containing an appropriate
chromogen can also be used to detect and determine other compounds
directly, such as bilirubin and pyruvic acid, without requiring
prior reaction with a substrate.
Chromogens which can be employed to react with the above compounds
to form colored reaction products are the well-known diazonium
salts of dyes, such as the water-soluble commercial fast color
salts. Illustrative examples are the diazonium salts of
6-benzamido-4-methoxy-m-toluidine (fast violet B, color index
37165), 5-nitro-2-aminoanisole-zinc chloride (fast red B, color
index 37125), 4'-amino-2',5'-diethoxybenzanilide (fast blue BB,
color index 37175), 4-benzoyl-amino-2,5-dimethoxyaniline (fast blue
RR, color index 37155) and the like. Even though these chromogens
are water soluble, they do not leach out of the test devices of the
present invention. The selectively permeable nature of the carrier
material allows the test fluid to pass into the test device but
prevents the chromogen or the reacted color compound from passing
out.
The preferred chromogen in the test devices for detection and/or
determination of enzymes is an insoluble polymeric diazonium salt
of an aromatic polyamine bound through an amide linkage to a cation
exchange resin. This chromogen is prepared by reacting a cation
exchange resin which contains acid groups, such as carboxylic
groups, with a halogenating agent, such as thionyl chloride, to
convert at least some of the acid groups of the resin to acyl
halide groups. The resulting acyl halide groups are then reacted
with an aromatic polyamine, such as o-dianisidine, to provide a
resin containing amide linkages and free aromatic amino groups.
This product is then treated by well-known techniques to diazotize
the free aromatic amino groups to produce the insoluble polymeric
diazonium salt. This product is insoluble in both aqueous and
nonaqueous systems.
The test devices of the present invention can also be used to
measure the pH of a fluid system. In these cases the test system
includes a pH-sensitive material as the chromogen. It is preferred
that the pH indicator material itself be insoluble. The insoluble
pH indicator can consist of the reaction product of an insoluble
polymeric material and a pH sensitive compound. Such insoluble
indicators can be prepared in various ways. An insoluble polymeric
diazonium salt of an aromatic polyamine bound through an amide
linkage to a cation exchange resin, described above, can be reacted
with a suitable coupling compound, such as N,N-dimethyl aniline.
The resulting reaction product undergoes color changes at various
pH values depending on the specific polyamine employed in the
polymeric diazonium salt.
Another form of pH indicator can be prepared by reacting a
well-known diazonium salt of para-aminobenzyl cellulose with a
suitable coupling compound, such as alpha-naphthylamine. The
resulting reaction product undergoes a color change from violet to
orange-red in the pH range from 4 to 6.
Still another form of pH indicator can be prepared by reacting a
soluble compound having at least one free amino or phenolic group,
such as rosaniline, with a polycarboxylic resin acyl halide. This
polycarboxylic resin acyl halide can be prepared by reacting a
cation exchange resin containing carboxylic acid groups with a
halogenating agent, such as thionyl chloride. The resulting
reaction product undergoes a color change when changing from acidic
to alkaline conditions.
Still a further form of pH indicator can be prepared by reacting a
suitable diazonium salt, such as the diazonium salt of
6-benzamido-4-methoxy-m-toluidine (fast violet B, color index
37165) with the reaction product of a polycarboxylic resin acyl
halide and a suitable aromatic compound having at least one free
amino group, such as N-(1-naphthyl)ethylene diamine.
The test devices of the present invention can also contain test
systems for other components, such as urea in blood. In this form
of the invention the test system comprises a urease enzyme which
catalyzes the hydrolysis of urea to liberate ammonia, a suitable
buffer and a pH indicator which changes color from yellow to blue
in the pH range of 5-8. A suitable pH indicator is the reaction
product of an ion exchange resin having free amino groups and
dibromothymolsulfonphthalein (bromthymol blue). Other pH indicators
useful in a teat for urea which change color from yellow to red in
the pH range of 6-8 are the reaction products of an ion exchange
resin having free amino groups and o-cresolsulfonphthalein (cresol
red) or phenolsulfonphthalein (phenol red) or
m-cresolsulfonphthalein (metacresol purple).
THe water-resistant carrier employed in the present invention to
contain the test system is preferably a colorless transparent
organoplastic material which is at least partially soluble or is
able to swell in an appropriate solvent. When a transparent carrier
is employed, the resulting test device forms a reasonably
transparent test area containing the chromogen. The colors
developed by the chromogen in the test area can then be read
visually or instrumentally under reflected or transmitted light
conditions. This is an advantage of the improved test device of the
present invention as compared to most prior art devices which can
be read only under reflected light conditions. In one form of the
invention a mixture of carrier material, solvent and test system is
cast as a film and allowed to solidify by evaporation of the
solvent. The resulting film can be self-supporting or it can be
bonded to a suitable supporting member. In a preferred form of the
invention the carrier material in solidified form is contacted with
a liquid mixture of solvent and test system. The solvent allows the
test system to penetrate into at least a surface portion of the
carrier. The solvent is then evaporated. The following are
illustrative examples of suitable water-resistant carrier materials
and appropriate solvents for use therewith.
---------------------------------------------------------------------------
Carrier Material Solvent
__________________________________________________________________________
Celluloid Acetone Methylethylketone Cellulose acetate Acetone
Acetone-methylethylketone mixture Ethyl cellulose Benzene
Polymethylmethacrylate Chloroform Carbon tetrachloride Polystyrene
Benzene Toluene Cellulose nitrate Acetone Polyvinyl chloride
Acetone Polyethylene Benzene
__________________________________________________________________________
If desired, various fillers, such as cellulose, barium sulfate,
alumina and the like can be included in the test device to add bulk
to the test system and to aid in color definition. These fillers
tend to increase the opacity of the test area of the test
device.
The invention will be described in further detail in the following
examples.
EXAMPLE 1
A polymethocrylic cation exchange resin in the acid form having
carboxylic acid reactive groups (Amberlite IRP 64) was milled into
a fine powder having a particle size range from 20 to 50 microns
and dried. A 50 g. portion of this powder was refluxed in 200 ml.
of thionyl chloride under stirring for 6 hours. The reaction
mixture was then filtered and the resulting chlorinated resin was
washed with anhydrous toluene and dried under vacuum. A 10 g.
portion of this chlorinated resin was poured into a solution of 3.3
g. of o-dianisidine in 75 ml. of dry toluene. The resulting
suspension was heated at 85.degree. C. for 5 hours with stirring.
The reaction mixture was filtered and the resin was washed with 100
ml. of ethanol from which it was possible to recover the unreacted
amine. The resin was then placed into a chromatographic column and
washed with 200 ml. of 2N HBF.sub.4 and then with water. A 3 g.
quantity of the thus-prepared resin-acylated amine was suspended in
10 ml. of 3N HBF.sub.4 and cooled to a temperature of 0.degree.
-5.degree. C. The resulting suspension was stirred while adding
dropwise 10 ml. of 1N NaNO.sub.2. Stirring was continued and the
temperature maintained for 4 hours. The mixture was then
centrifuged and the isolated product was dried in a vacuum. This
product had the formula of Resin-- CO-- NH-- Ar-- N.sup.+ N--
BF.sub.4, wherein "Resin" represents the cation exchange resin and
"Ar" represents the dianisidine nucleus. This product is an
insoluble polymeric diazonium salt of an aromatic polyamine bound
through an amide linkage to a cation exchange resin. This product
was further milled to a fine powder having particle sizes in the
range of 1-5 microns.
A mixture of 0.2 g. of the above product, 0.015 g. cellulose
acetate, 2 ml. acetone and 2 ml. methylethylketone was homogenized.
A 0.2 mm. thick strip of celluloid, 5 mm. wide and 66 mm. long, was
treated with a portion of the above liquid mixture, then dried in
air and under vacuum. The resulting strip product consisted of a
water-resistant celluloid organoplastic carrier containing, as an
integral part thereof, the above insoluble polymeric diazonium salt
of an aromatic polyamine bound through an amide linkage to a cation
exchange resin. This chromogen is capable of forming colored
reaction products with various coupling compounds and thus acting
as a test system.
The above-prepared test device had a yellow color in the area
containing the chromogen. When this test device was placed in an
aqueous alkaline solution containing 350 micrograms of
beta-naphthol per ml. of solution, the test device turned to a
red-violet color indicating reaction between the beta-naphthol and
the chromogen.
EXAMPLE 2
The procedure of example 1 was repeated several times using as the
aromatic polyamine benzidine, o-tolidine,
4,4'-diaminodiphenylamine, m-phenylenediamine,
1,8-diaminoaphthalene, 2-Cl-1,4-diaminobenzene, 2-NO.sub.2
-1,4-diaminobenzene, and 2-SO.sub.3 H-1,4-diaminobenzene to form
different chromogens. These chromogens were then employed
individually in the cellulose acetate, acetone, methylethylketone
mixture of example 1 to treat celluloid strips in the manner
described in example 1. The resulting test devices were then placed
in aqueous alkaline solutions containing 350 micrograms of
beta-naphthol per ml. of solution and color changes were noted. The
following table shows the color changes indicating reaction between
the beta-naphthol and the chromogen contained within the celluloid
carrier.
---------------------------------------------------------------------------
Amine Constituent Test Device Test Device of Color Developer
Initial Color Final Color
__________________________________________________________________________
Benzidine Yellow Red o-tolidine Yellow Violet
4,4'-diaminodiphenylamine Green Brown m-phenylenediamine Yellow
Orange-Red 1,8-diaminonaphthalene Orange Red-Brown 2-Cl-1,4
-diaminobenzene White Red 2 -NO.sub.2 -1,4 -diaminobenzene Yellow
Red 2 -SO.sub.3 H-1,4-diaminobenzene White Red
__________________________________________________________________________
The above examples all employed water-insoluble chromogens. The
following example describes use of a water-soluble test system
contained in a water-resistant carrier.
EXAMPLE 3
A mixture of 0.025 g. of the diazonium salt of
6-benzamido-4-methoxy-m-toluidine (fast violet B, color index
37165), 0.020 g. cellulose acetate, 0.008 g. HBF.sub.4, 4 ml.
acetone and 0.010 g. TiO.sub.2 was homogenized. A 0.2 mm. thick
strip of celluloid, 5 mm. wide and 60 mm. long, was treated with a
portion of the above liquid mixture, then dried in air and under
vacuum. The resulting test device product containing the diazonium
salt within the celluloid carrier was then placed in an aqueous
alkaline solution containing up to 500 micrograms of beta-naphthol
per ml. of solution. The strip color changed from violet to
red-violet indicating reaction between the diazonium salt and the
beta-naphthol. The water-soluble diazonium salt did not leach out
of the celluloid strip indicating that it was embedded within the
celluloid.
EXAMPLE 4
The procedure of example 3 was repeated using different
water-soluble diazonium salts. The resulting celluloid strips
containing the diazonium salts where then immersed in aqueous
alkaline solutions of beta-naphthol and color changes were
observed. The water-soluble salts did not leach out of the
celluloid. The following table shows the color changes for the
diazonium salts employed.
---------------------------------------------------------------------------
Original Final Diazonium Salt of Color Color
__________________________________________________________________________
5-nitro-2-aminoanisole-ZnC1.sub.2 Red Bright red (Fast Red B, Color
Index 37125)
4'-amino-2', 5'-diethoxybenzanilide Blue Red-violet (Fast Blue BB,
Color Index 37175) 4-benzoyl-amino- 2,5-dimethoxy- Blue Red-violet
aniline (Fast Blue RR, Color Index 37155)
__________________________________________________________________________
The test devices prepared in examples 1-4 are useful to
qualitatively detect and/or quantitatively determine beta-naphthol.
The amount of color change in the chromogen is directly related to
the amount of beta-naphthol present in the test sample. This
reaction can also be used to detect and/or measure phosphatase
enzymes. If a test sample containing a phosphatase is treated with
a suitable buffer and a beta-naphthyl phosphate, the enzyme will
liberate beta-naphthol which can then be detected and measured by
the above test devices. It is also possible to include the buffer
and the beta-naphthyl phosphate in the test device so that the
phosphatase analysis can be simplified. This is described in the
following example.
EXAMPLE 5
The procedure of example 1 was repeated employing 20 mg. of
beta-naphthyl phosphate in the liquid mixture used to treat the
celluloid strip. Only one side of the celluloid strip was treated
with this mixture for a length of 5 mm. The other side of the strip
was masked by an adhesive band which was removed after the strip
had been dried. A strip of filter paper 5 mm. wide was then
immersed in a 50 weight percent aqueous solution of tris
(hydroxymethyl) aminomethane buffer and dried. It was then cut into
segments 5 mm. long. One such segment was attached by adhesive to
the side of the above celluloid strip which had previously been
masked. The resulting test device having a 5 mm..times. 5 mm. test
area was then immersed in a 0.5 ml. test sample of serum containing
a known amount of alkaline phosphatase for 5 minutes at 37.degree.
C. The tris (hydroxymethyl) aminomethane buffer was leached out of
the filter paper by the serum and provided a sample pH of 10-12.
These alkaline conditions are necessary for detection of an
alkaline phosphatase. The alkaline phosphatase in the serum entered
the test device and reacted with the beta-naphthyl phosphate. The
beta-naphthol liberated from the beta-naphthyl phosphate by the
alkaline phosphatase then caused a color change in the test device
chromogen contained within the celluloid strip. The resulting color
was compared to a color chart having a correlation between color
and alkaline phosphatase concentration to determine the alkaline
phosphatase content of the test sample. The alkaline phosphatase
content thus determined corresponded to the known amount of
alkaline phosphatase in the serum.
EXAMPLE 6
The procedure of example 5 was repeated employing a filter paper
segment impregnated with an aqueous solution of sodium citrate
instead of the tris (hydroxymethyl) aminomethane. The resulting
test device was then immersed in a 0.5 ml. test sample of serum
containing a known amount of acid phosphatase for 10 minutes at
37.degree. C. The sodium citrate buffer provided a sample pH of
4.5. The beta-naphthol liberated by the acid phosphatase then
caused a color change in the test device which was compared to an
acid phosphatase color chart to determine the acid phosphatase
content of the test sample. The acid phosphatase content thus
determined corresponded to the known amount of acid phosphatase in
the serum.
Example 7
A mixture of 200 mg. of a chromogen consisting of diazotized
o-dianisidine linked by amidic coupling to a carboxylic cation
exchange resin prepared by the method of example 1, 40 mg. of ethyl
cellulose, 100 mg. of barium sulfate, 2 ml. of benzene and 2 ml. of
acetone was thoroughly agitated. A strip of polystyrene 0.2 in.
wide, 3 in. long and 0.2 mm. thick was dipped into the above
mixture for 5 seconds and then dried at room temperature. The above
mixture components were incorporated in a plastic matrix since they
appeared to be covered by a plastic film. This reagent strip was
then immersed in a saturated alkaline solution of beta-naphthol for
10 minutes during which time a light brown color developed in the
strip indicating reaction between the chromogen and the
beta-naphthol.
EXAMPLE 8
Strips of cellulose nitrate and cellulose acetate were dipped in
the above-described mixture of example 7 for three seconds and
dried at room temperature. The mixture components appeared to be
incorporated in the strips. These strips were then immersed in a
saturated alkaline solution of beta-naphthol and a red-brown color
developed indicating reaction between the chromogen and the
beta-naphthol.
EXAMPLE 9
A paper strip was dipped in a benzene suspension of polyethylene
for a few seconds and then cured in a 105.degree. C. oven for 30
minutes. The resulting polyethylene-coated paper strip was then
dipped in the above-described mixture of example 7 for 10 seconds
and dried at room temperature. The chromogen mixture appeared to
form a coating along the strip and to be partially embedded in the
polyethylene. This reagent strip then produced a positive color
reaction when contracted with an alkaline beta-naphthol
solution.
EXAMPLE 10
A mixture of 200 mg. of the chromogen of example 7, 200 mg. of
barium sulfate, 40 mg. of cellulose acetate, and 4 ml. of acetone
was thoroughly agitated. A strip of polyvinylchloride was dipped
into the above mixture and then dried at room temperature. This
reagent strip containing a chromogen incorporated in a plastic
matrix produced a positive color reaction when contacted with an
alkaline beta-naphthol solution.
The above examples all formed the test device by treating the
water-resistant carrier member with a solvent-containing chromogen
mixture wherein the solvent acts on the carrier to partially
dissolve at least a surface layer of the carrier to allow the
chromogen to become embedded in the carrier. The following example
describes the formation of test devices having a water-resistant
carrier containing a chromogen wherein the carrier forms a film
adherent to a base member of different composition.
EXAMPLE 11
A 200 mg. portion of the chromogen described in example 7 was mixed
with a 1 percent (weight/volume basis) solution of cellulose
acetate in acetone to form a total volume of 3.5 ml. The following
base materials were dipped in the above mixture: filter paper,
wood, aluminum foil, glass, stainless steel, and
polytetrafluoroethylene. The so-treated materials were then dried
at room temperature. A water-resistant cellulose acetate coating
containing the chromogen was formed on the surface of each of the
above materials. These coated base materials were then contacted
with an alkaline solution of beta-naphthol and they all developed
red colors within 60 seconds indicating reaction between the
chromogen and the beta-naphthol.
The above examples all related to the detection of beta naphthol
and especially the detection and/or determination of beta-naphthol
liberated from beta-naphthyl phosphate by the action of alkaline
phosphatase or acid phosphatase. The test devices of the present
invention can also be used for the detection and/or determination
of other substances as described in the following examples.
EXAMPLE 12
A 30 mg. portion of the diazonium salt of 5-nitro-2-amino-toluene
(fast red RL, color index 37100) chromogen was dissolved in a
mixture of 2 ml. acetone and 10 mg. cellulose acetate. A strip of
celluloid was treated with this solution and dried in air and then
under vacuum. The resulting test device had a water-resistant film
containing the chromogen and had a yellow color in the chromogen
area. This test device was then immersed in an aqueous alkaline
solution containing 500 micrograms of alpha-naphthol per ml. of
solution. This solution also contained 1.5 M tris (hydroxymethyl)
aminomethane buffer. The reactive portion of the test device turned
an orange color indicating reaction with the alpha-naphthol.
EXAMPLE 13
A celluloid test device containing the insoluble polymeric
diazonium salt of 2-Cl-1,4 -diaminobenzene bound through an amide
linkage to a cation exchange resin was prepared by the technique
described in example 2. This test device has a white color in the
reactive zone. A 0.1 ml. portion of an aqueous solution of 0.2 M
sodium aspartate, 0.1 M alpha-ketoglutarate and 0.1 M phosphate
buffer at pH 7.4 was placed in a test tube. To this was added 0.2
ml. of a test serum containing glutamic oxaloacetic transaminase.
This mixture was maintained at 37.degree. C. for 20-30 minutes. The
above test device was then immersed in the reaction solution for 2
minutes. The reactive zone of the test device turned orange-red
indicating reaction between the chromogen in the test device and
the oxaloacetic acid liberated by the transaminase from the
alpha-ketoglutarate-sodium aspartate substrate.
EXAMPLE 14
A celluloid test device containing the insoluble polymeric
diazonium salt of 2-NO.sub. 2 -1,4 -diaminobenzene boutnd through
an amide linkage to a cation exchange resin was prepared by the
technique described in example 2. A 0.5 ml. portion of an aqueous
solution of 6.85 .times.10.sup..sup.-4 M
alpha-leucine-beta-naphthylamine hydrochloride substrate and 0.2 M
phosphate buffer at pH 7 was placed in a test tube. To this was
added 0.05 ml. of a test serum containing leucine aminopeptidase.
This mixture was maintained at 37.degree. C. for one hour. The
above test device was then immersed in the reaction solution for 5
minutes. The reactive zone of the test device turned red-violet
indicating reaction between the chromogen and the
beta-naphthylamine liberated by the enzyme in the serum from the
substrate.
EXAMPLE 15
A celluloid test device of the type described in example 1 having a
yellow reactive zone was employed in this example. A 1ml. portion
of an aqueous solution of 0.2 mg. beta-naphthyl-laurate substrate
per ml. of solution, 0.1 M tris (hydroxymethyl)-aminomethane buffer
and 20 weight percent sodium cholate was placed in a test tube. To
this was added 0.1 ml. of a test serum having lipase activity. This
mixture was maintained at 37.degree. C. for 30 minutes. The above
test device was then immersed in the reaction solution for 5
minutes. The reactive zone of the test device turned red-violet
indicating reaction between the chromogen and the beta-naphthol
liberated from the substrate by the lipase.
EXAMPLE 16
A cellulose acetate strip was placed in a cupric chloride-acetone
solution for a few minutes. The so-treated strip was then removed
from the solution, and the acetone was allowed to evaporate. The
strip was then placed in a neocuproine-acetone solution for a few
minutes and removed. After the acetone evaporated, a smooth green
colored strip resulted which reacted quantitatively with various
levels of ascorbic acid to change the color of the strip to various
shades of amber-orange corresponding to the amount of ascorbic acid
present.
The above examples describe the use of a test system incorporated
in a water-resistant carrier to react with an active ingredient of
a test fluid in order to detect or determine some component of the
fluid system. The present invention also includes test devices
useful for measurement of pH wherein the component or
characteristic of the fluid system being detected or determined is
the hydrogen ion concentration or pH. This is described in the
following examples.
EXAMPLE 17
An insoluble polymeric diazonium salt of benzidine bound through an
amide linkage to a carboxylic cation exchange resin was prepared in
accordance with the techniques of examples 1 and 2. A 5 g. portion
of this product was then suspended in 20 ml. of a buffer at pH 5
consisting of a mixture of tris(hydroxymethyl aminomethane and 2 M
acetic acid. To this suspension at 20.degree. C. was added with
stirring a 50 percent stoichiometric excess of N,N-dimethylaniline.
The stirring was continued for 20 minutes to form an insoluble
indicator which is the reaction product between the above diazonium
salt and the N,N-dimethyl-aniline coupling compound. This indicator
was then recovered by filtration, washed with water and dried. A
mixture was then prepared consisting of 400 mg. of the
above-prepared indicator, 400 mg. cellulose powder, 80 mg.
ethylcellulose, 2 ml. benzene and 2 ml. acetone. A celluloid strip
was immersed in the above liquid mixture and then dried. The
resulting product was a celluloid strip containing the above
indicator within the strip. When placed in an aqueous medium having
a pH of 1, the test strip had a violet color. When placed in an
aqueous medium having a pH of 2, the test strip had a yellow color.
This color change is reversible when the medium pH being measured
varies through this range.
EXAMPLE 18
Insoluble polymeric diazonium salts of aromatic polyamines bound
through an amide linkage to a carboxylic cation exchange resin were
prepared using several different aromatic polyamines according to
the technique of example 1. These diazonium salts were then reacted
with various coupling compounds according to the technique of
example 17 to form insoluble indicators. These indicators were then
incorporated into celluloid strips according to the technique of
example 17. The resulting indicator test devices were then employed
to detect pH changes. The following are the characteristics of the
test devices so produced:
---------------------------------------------------------------------------
Aromatic Polyamine Coupling Compound pH Color
__________________________________________________________________________
m-phenylenediamine N,N-dimethylaniline 1 Red 2 Yellow
1,4-diamino- 6-benzene N,N-dimethylaniline 3 Red -sulfonic acid 4
Yellow 2-chloro- 1,4-phenylene N,N-dimethylaniline 1 Violet
-diamine 2 Yellow
2-chloro-1,4 -phenylene 8-hydroxyquinoline 12 Brown -diamine 13
Purple
__________________________________________________________________________
EXAMPLE 19
Powdered cellulose was thoroughly washed with dilute acid, dilute
alkali, and water, and then dried. A 4 g. portion of this material
was then mixed with 12 g. of p-nitrobenzylchloride and 30 ml. of 40
weight percent aqueous sodium hydroxide and stirred vigorously at
95.degree. C. The reacting mixture was cooled during the first part
of the exothermic reaction. After 4 hours the mixture was poured
into a large excess of cold water and filtered. The residue was
then washed with water, with ethanol and finally with acetone. A 5
g. portion of the above-prepared p-nitrobenzylcellulose was
suspended in 50 ml. of ethanol and heated to near boiling. The
mixture was then stirred vigorously and 5 g. of sodium hydrosulfite
dissolved in water was slowly added. After continued heating of the
mixture for about 30 min., the light yellow product was filtered
off and washed with cold water. A 5 g. portion of the
above-prepared p-aminobenzylcellulose was suspended in 10 ml. of 2
N hydrochloric acid, then mixed with 20 ml. of water and chilled in
an ice bath. With constant stirring, 0.5 M sodium nitrite solution
was slowly added until a test with potassium iodide-starch paper
remained positive for 15 min. after addition of the last portion of
nitrite. Stirring was then continued for another 15 minutes, and
the material was filtered and washed with weakly acid ice water.
This product is the diazonium salt of p-aminobenzylcellulose. This
diazonium salt was then reacted with various coupling agents
according to the technique of example 17 to form insoluble
indicators. These indicators were then incorporated into celluloid
strips by treating the celluloid with a mixture of 800 mg. of the
indicator, 80 mg. of ethyl cellulose, 2 ml. benzene and 2 ml.
acetone. The resulting indicator test devices were then employed to
detect pH changes. The following are the characteristics of the
test devices so produced:
---------------------------------------------------------------------------
Coupling Compound pH Color
__________________________________________________________________________
N-(1 -naphthyl)ethylenediamine 2-3 Violet 3-4 Orange-red
Alpha-naphthylamine 4-5 Violet 5-6 Orange-red
1,8-diaminonaphthalene 5 Green-blue 6 Violet-red
8-hydroxyquinoline 12 Yellow 13 Red
__________________________________________________________________________
EXAMPLE 20
A 5 g. portion of a polycarboxylic resin acyl chloride prepared in
accordance with the technique of example 1 was added with stirring
to an 80.degree. C. toluene solution of basic rosaniline. A 10 ml.
amount of triethylamine was then added dropwise and the stirring
and heating were continued for 3 hours. The resulting product was
then filtered and washed. This is an insoluble indicator
represented by the reaction product of the resin acyl chloride and
rosaniline. This indicator was then incorporated into a celluloid
strip according to the technique of example 17. When placed in an
acid solution, the resulting test device had a medium pink color.
When placed in an alkaline solution, the resulting test device had
a dark pink-violet color.
EXAMPLE 21
A polycarboxylic resin acyl chloride prepared in accordance with
the technique of example 1 was reacted with N-(1-naphthyl)
ethylenediamine in accordance with the procedure of example 20. A 5
g. portion of the resulting product was dispersed in 40 ml. of a pH
5 buffer solution of acetic acid and tris (hydroxymethyl)
aminomethane. To this suspension was added with stirring at room
temperature 0.1 g. of a suitable diazonium salt. The stirring was
continued for 20 minutes and the reaction product was then filtered
and washed. The resulting indicator was then incorporated in a
celluloid strip according to the technique of example 17. This
procedure was repeated for several different diazonium salts. The
resulting indicator test devices were then employed to detect pH
changes. The following are the characteristics of the test devices
so produced.
---------------------------------------------------------------------------
Diazonium Salt of pH Color
__________________________________________________________________________
6-benzamido- 4-methoxy-m-toluidine 1 Blue (fast violet, Color Index
37165) 2 Red
Sulfanilic acid 12 Violet 13 Red o-Dianisidine 1 Blue 2 Violet
p-Amino-N,N-dimethylaniline 1 Violet 2 Yellow
__________________________________________________________________________
EXAMPLE 22
A 2 g. portion of a polystyrene weakly basic ion exchange resin
having free amino groups (Amberlite IR-45) was suspended in a
mixture of 50 ml. ethanol and 50 ml. water at room temperature. To
this were added 100 mg. of dibromothymolsulfonphthalein (bromthymol
blue) and the resulting mixture was reacted for 3 hours under
reflux conditions. The resulting resin-indicator complex was washed
with water and with 0.2 N HCl and 0.2 N NaOH and then dried. A 200
mg. portion of the above product was mixed with 200 mg. cellulose
powder, 20 mg. KH.sub.2 PO.sub.4 buffer, 50 mg. urease, and 4 ml.
of a 2 percent solution of ethylcellulose in acetone also
containing 0.1 percent polyoxyethylene lauryl ether surfactant
(BRIJ). Strips of polystyrene, cellulose acetate and celluloid 5
mm. wide, 60 mm. long and 0.2 mm. thick were treated with the above
liquid mixture for a length of 5 mm. and dried. The 5 mm. .times. 5
mm. test areas of these strips were then immersed for 2-3minutes in
different 0.5 ml. liquid serum samples containing various amounts
of urea from 20 mg./100 ml. to 120 mg./100 ml. The color of the
indicator in the test strip had reproducible differences for
different levels of urea and can thus be used to quantitatively
measure the urea content of serum.
EXAMPLE 23
A 1 g. portion of the diethylaminoethyl modified form of a
polysaccharide dextran suitably cross-linked with epichlorohydrin
to produce a hydrophilic solid gel characterized by a high degree
of microporosity and containing reactive amino groups
(DEAE-Sephadex) was reacted with 0.08 g. of bromthymol blue
according to the procedure of example 22. A 200 mg. portion of the
resulting indicator complex was mixed with 250 mg. cellulose powder
containing 20 weight percent adsorbed urease, 20 mg. of KH.sub.2
PO.sub.4, and 4 ml. of a 2 percent solution of ethylcellulose in
acetone also containing 0.2 percent of a polyoxyalkylene derivative
of sorbitan monolaurate (Tween 20). Strips of polystyrene were
treated with the above liquid mixture and dried. They were then
dipped into a 1 percent solution of ethylcellulose in acetone and
dried. The resulting test devices were then immersed for 2 min. in
different liquid serum samples containing various amounts of urea
from 20 mg./100 ml. to 120 mg./100 ml. The color of the indicator
in the test strip had reproducible differences for different levels
of urea in the test samples.
The above-described test devices for detection of urea can also be
employed to detect urea in blood. The test device is contacted with
a blood sample for an appropriate period of time and then the blood
is wiped or washed off the test device. The resulting color in the
reaction portion of the test device is then compared with a color
chart showing the relationship between color and urea content to
determine the amount of urea in the blood sample. The
water-resistant carrier of the test device acts as a selectively
permeable membrane to prevent the red blood cells and interfering
proteins of the blood sample from penetrating into the test system
and interfering with the color formation.
* * * * *